Deinococcus Radiodurans  the Consummate Survivor

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DEINOCOCCUS RADIODURANS  THE CONSUMMATE SURVIVOR

Michael M. Cox* and John R. Battista‡ Abstract | Relatively little is known about the biochemical basis of the capacity of Deinococcus radiodurans to endure the genetic insult that results from exposure to ionizing radiation and can include hundreds of DNA double-strand breaks. However, recent reports indicate that this species compensates for extensive DNA damage through adaptations that allow cells to avoid the potentially detrimental effects of DNA strand breaks. It seems that D. radiodurans uses mechanisms that limit DNA degradation and that restrict the diffusion of DNA fragments that are produced following irradiation, to preserve genetic integrity. These mechanisms also increase the efficiency of the DNA-repair proteins.

1 IONIZING RADIATION In 1956, Anderson et al. isolated a novel vegetative of genetic tools. Evidence has accumulated for both Any electromagnetic or bacterium from canned ground meat that had been passive and enzymatic contributions to genome res- particulate radiation powerful γ-irradiated at 4,000 Gray (Gy), a dose that is approx- titution (FIG. 1), which provides a framework for our enough to strip electrons from imately 250 times higher than that typically used to discussion in this review. atoms to produce ions. kill Escherichia coli. The authors named this species Micrococcus radiodurans because of its superficial Ionizing-radiation resistance morphological similarity to members of the genus Of all the phenotypes associated with prokaryotes, Micrococcus. However, research on M. radiodurans resistance to ionizing radiation is one of the most dif- over the next 30 years resulted in reclassification of ficult to rationalize in terms of natural selection. As this species and its closest relatives into a distinct there are no naturally occurring environments known phylum within the domain Bacteria2–7. The genus that result in exposures exceeding 400 mGy per year9, name — Deinococcus — was based on the Greek it is unlikely that species evolved mechanisms to adjective ‘deinos’, which means strange or unusual8; protect themselves against the effects of high-dose an apt description for an organism with an ability to ionizing radiation per se. Instead, it seems that the survive genetic damage that sets it apart from much damage introduced by γ-irradiation shares features of the life on Earth. with the damage that results from other stresses to Treatment of Deinococcus radiodurans with high which bacteria have adapted. For example, desicca- *Department of levels of IONIZING RADIATION can produce hundreds tion introduces many DNA double-strand breaks into Biochemistry, University of genomic double-strand breaks TABLE 1, but the the genomes of D. radiodurans10 and members of the of Wisconsin–Madison, genome is reassembled accurately before initiation cyanobacterial genus Chroococcidiopsis11. Both organ- Wisconsin 53706-1544, of the next cycle of cell division. The extraordinary isms are tolerant to desiccation and are resistant to the USA. ‡Department of Biological capacity of Deinococcus spp. to reconstitute their potentially lethal effects of ionizing radiation, which Sciences, Louisiana State genomes has inspired a small but growing commu- might indicate that the radioresistance of these species University and A&M nity of researchers who are interested in the relevant is a fortuitous consequence of their ability to tolerate College, Baton Rouge, mechanisms that are used to achieve this. In recent desiccation-induced strand breaks. Louisiana 70803, USA. Correspondence to J.R.B. years, investigation of the biology and biochemistry of TABLE 2 lists members of six bacterial phyla that are e-mail: [email protected] Deinococcus spp. has accelerated, catalysed by the avail- resistant to ionizing radiation. The Deinococcaceae doi:10.1038/nrmicro1264 ability of genome information and the development are the best-known family on this list, and have been

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Table 1 | Double-strand breaks formed in E. coli and D. radiodurans is possible that these diverse prokaryotic cells adapted differently to a similar selective pressure and that there Species* Genomes DNA double-strand Average distance per cell breaks‡ at D between lesions might be multiple mechanisms of radioresistance. 37 This latter possibility seems more probable, as fewer Escherichia coli K12 4–5 8–9 530,000 bp independent evolutionary events (the loss of resistance Deinococcus 8–10 275 10,000 bp from each species being considered a separate event) radiodurans R1 would be required to generate the handful of known *In each study, actively growing aerobic cultures were irradiated and the number of double-strand radioresistant species. breaks determined using neutral sucrose-gradient centrifugation. ‡For D. radiodurans this is lesions per unit length of chromosome I (2.64 Mb). Chromosome I is used because it is the largest target Radiation resistance is not restricted to the domain within the cell. Calculations are based on a measured rate of 1 DNA double-strand break per 10 Bacteria. Several HYPERTHERMOPHILIC archaea (members Gray per 5×109 Da of double-stranded DNA. D , the dose at which 37% of the cells survive. 37 of the Euryarchaeota and Crenarchaeota) show extreme ionizing-radiation resistance12–15, in some cases (for studied for five decades. Little has been learnt about the example, Thermococcus gammatolerans) comparable other bacterial species listed, other than their capacity to to that of Deinococcus radiodurans14. tolerate ionizing radiation. Draft genome sequences of Rubrobacter xylanophilus and Kineococcus radiotolerans The Deinococcaceae have been released in the past year, but detailed analyses The Deinococcaceae family comprises 11 validly of these sequences have not yet been reported. described species — D. radiodurans, Deinococcus There is no clear pattern of evolution among proteolyticus, Deinococcus radiopugnans, Deinococcus ionizing-radiation-resistant species (FIG. 2). The scat- grandis, Deinococcus radiophilus, Deinococcus geo- tered appearance of ionizing-radiation resistance thermalis, Deinococcus murrayi, Deinococcus indi- among distinct prokaryotic lineages indicates two cus, Deinococcus frigens, Deinococcus saxicola and possibilities. First, radioresistance could be a ves- Deinococcus marmoris — all grouped in a single genus, tige of DNA-repair mechanisms that were present Deinococcus16–18. Deinococci do not form spores and in ancestral species and have been retained in those are non-motile. Most species grow best in rich media organisms that continue to require this phenotype. at temperatures between 30 and 37oC, with a doubling This explanation assumes that the ancestor’s ability time between 1.5 and 3 hours. However, D. geotherma- to cope with DNA damage has been lost by most lis and D. murrayi are true thermophiles, with optimal descendents, and predicts that the molecular mecha- growth temperatures of 45–55oC. With the exception nisms of radioresistance should be similar among of D. grandis, which is rod-shaped, all Deinococcus spe- ionizing-radiation-resistant species. Second, given cies are spherical cells that exist singly or as pairs and the infrequent occurrence of ionizing-radiation resist- tetrads in liquid culture. ance, it is possible that this phenotype has arisen in To date, D. radiodurans has received more attention unrelated species through horizontal gene transfer, or than the other deinococci. The genome of D. radiodurans possibly convergent evolution. Much as birds and bats strain R1 (ATCC BAA-816) has been sequenced19,20 and evolved wings despite distinct evolutionary origins, it can be accessed at the TIGR Comprehensive Microbial Resource database (see Online links box). The D. radiodurans chromosome is 3.28 Mb, with a GC content of

Ionizing radiation 66.6%. There are nine types of short nucleotide repeats, ranging in size from 60 to 215 bp, found at 295 sites DNA double- strand breaks that are randomly scattered in the genome. The genome Mn Mn Mn Mn is segmented and consists of a 2.64-Mb chromosome (chromosome I), a 0.41-Mb chromosome (chromosome II), a 0.18-Mb megaplasmid and a 0.045-Mb plasmid21. D. radiodurans has between 4 and 10 genome copies per cell, depending on the stage of the bacterial growth phase22,23. γ Mn Mn Repair Mn Mn The -irradiation survival curves of actively grow- Mn ing cultures of D. radiodurans R1 have a shoulder Mn Mn Mn of resistance up to 5,000 Gy24, and until this dose is achieved there is no measurable loss of viability in the

irradiated culture. Under these conditions, the D37 dose BOX 1 for D. radiodurans R1 is approximately 6,500 Gy. Assuming that there are eight genome copies per cell in these cultures, a 5,000-Gy dose will introduce Mn Mn Mn Mn approximately 1,600 double-strand breaks per cell. As this dose is sublethal, it can be inferred that potentially Figure 1 | Potential contributions to the recovery from radiation damage in Deinococcus radiodurans. The schematic depicts a D. radiodurans tetracoccus. catastrophic deletions and genome rearrangements The nucleoid in each compartment is highly condensed and maintains its overall architecture occur at low frequencies. Although there is no formal after irradiation. High levels of Mn(II) might contribute to the recovery from DNA damage. proof, it seems that the process of double-strand-break A wide range of enzymes probably also contribute to genome reconstitution. repair in D. radiodurans is error-free.

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Table 2 | Species of ionizing-radiation-resistant bacteria mechanism that blocks the formation of strand breaks in the D. radiodurans genome unlikely. In contrast Species Representative Phylum Refs D value* to most other cells, D. radiodurans has the ability to 10 efficiently and accurately repair that damage. α Methylobacterium 1,000 Gray -Proteobacteria 80,81 The observation that D. radiodurans cannot pas- radiotolerans sively prevent DNA damage does not rule out the Kocuria rosea 2,000 Gray Actinobacteria 4,8 possibility that other passive mechanisms contribute Acinetobacter radioresistens 2,000 Gray γ-Proteobacteria 82 to the capacity of the cell to tolerate DNA damage. Kineococcus radiotolerans 2,000 Gray Actinobacteria 83 Experimental evidence indicates that the recovery of D. radiodurans from substantial DNA damage relies on Hymenobacter actinosclerus 3,500 Gray Flexibacter–Cytophaga– 84 both passive features of deinococcal physiology and a Bacteroides robust complement of repair enzymes. There are sev- Chroococcidiopsis spp. 4,000 Gray Cyanobacteria 11 eral mechanisms that have the potential to contribute Rubrobacter xylanophilus 5,500 Gray Actinobacteria 85 to ionizing-radiation resistance. For example, there is Deinococcus radiodurans R1 10,000 Gray Deinococcus–Thermus 86 a clear requirement for RecA-dependent homologous recombination — this process has been examined and Rubrobacter radiotolerans 11,000 Gray Actinobacteria 85 reviewed extensively29–36. *The D defines the dose that is needed to eradicate 90% of the irradiated population. The D 10 10 In this review, we do not attempt to attribute the values for a given species vary substantially depending on growth conditions. The values provided are for actively growing oxygenated cultures. contribution to recovery from DNA damage that is made by each different mechanism, and the reader should bear in mind that new recovery mechanisms TABLE 1 compares the number of double-strand might still await discovery. DNA breaks that occur in the large chromosome of D. radiodurans R1 REF. 25 and the E. coli K12 REF. 26 Passive contributions to radioresistance

genome at the D37 radiation dose. The difference is Genome copy number. Cells with increased numbers striking: compared with E. coli K12, D. radiodurans tol- of genome copies have enhanced resistance to ioniz- erates approximately 30-fold more DNA double-strand ing radiation26,37. The extra genetic material is thought breaks before succumbing to the damage. Clearly, to protect the cell in two ways. First, when multiple D. radiodurans R1 cells have mechanisms to avoid the genomes are present, there are additional copies of cru- lethal effects of double-strand breaks that are absent cial loci that improve the chance of the cell surviving in E. coli. irradiation. The probability of inactivating a specific Whatever the mechanisms that D. radiodurans uses, gene in an organism is given by P=1–(1–1/M)L (where they do not prevent DNA damage. Recent evidence M is the total number of genes present, and L is the from two groups of researchers has shown that meas- number of inactivating lesions per genome). For a spe- urable double-strand breaks form at the same rate in cific dose of radiation, the probability of inactivating all E. coli and D. radiodurans if cultures are irradiated under the copies of a specific gene is equivalent to PN, where identical conditions27,28, which makes the existence of a N is the number of gene copies present (assuming that

Streptomyces albus Kineococcus radiotolerans Thermus aquaticus Kocuria Rubrobacter radiotolerans Chlorobium rosea limicola Deinococcus radiodurans

Chloroflexus Flexibacter aggregans aurantiacus Hymenobacter actinosclerus

Cytophaga hutchinsonii Planctomyces limnophilus

Thermotoga maritima

Acinetobacter radioresistens

Anabaena variabilis Escherichia coli Chroococcidiopsis thermalis Burkholderia cepacia Methylobacterium radiotolerans Rhodobacter capsulatus Spirochaeta aurantia Bdellovibrio bacteriovorans 0.10 Staphylococcus aureus Clostridium butyricum

HYPERTHERMOPHILIC Figure 2 | A 16S-rRNA-gene-sequence-based phylogeny of the main lineages of the domain Bacteria. The branches in Organisms that have an optimal red are those in which ionizing-radiation-resistant taxa have been described. The scale bar represents 10 inferred nucleotide growth temperature above 80°C. substitutions per 100 nucleotides.

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Box 1 | High-energy radiation and its effect on DNA Ionizing radiation is radiation with sufficient energy to ionize molecules. There are two types of ionizing radiation, both produced by the decay of radioactive elements: electromagnetic (X- and γ-radiation that form part of the electromagnetic spectrum that includes visible light and radio waves) and particulate (α- and β-particles). Different types of ionizing radiation deposit energy in matter at different rates. α- and β-particles produce ionization by collisions, depositing their energy within a short range after entering matter. γ-rays are photons that generate ions by several types of energy-absorption events (most commonly by the Compton effect, an increase in the wavelength of electromagnetic radiation when it collides with electrons in matter, see the figure) and can penetrate deeply into a cell or tissue. Ion production is accompanied by the release of energetic electrons (see the figure), and multiple ions and electrons can be generated in one event. The figure shows the tracks of three different types of ionizing radiation. Small dots indicate energy deposition events. The inset depicts the ejection of an electron from an atom to generate an ion, mediated by an encounter with a γ-ray photon. The γ-ray transfers part of its energy to a valance electron, which is thereby ejected from the nucleus to create an ion. The scattered γ-ray can undergo additional Compton effects within the matter. Ions react with other molecules to produce free radicals on a nanosecond timescale. In water, the products include highly reactive hydroxyl radicals. Typically, each energy deposition event can generate 2–5 radical pairs within a radius of a few nm. The energy deposited (the dose absorbed by the cell, tissue or other matter) is measured in rads or in Gray

(SI measurement; 100 rad = 1 Gray). The radioresistance of microorganisms is compared by measurement of the D37

dose (the dose at which 37% of the cells survive). At the D37 dose, each cell on average has experienced a lethal event (those that survive are balanced by other cells with two or more lethal events). The ions and free radicals produced as radiation passes through matter react rapidly and modify molecules. Of all the effects, genome damage probably has the greatest impact on cell viability. The reasons for this are threefold. First, genomic DNA occupies the largest fraction of the cell volume, and will therefore be ‘hit’ most often. Second, the genome is present in lower copy numbers than other molecules — there is little redundancy. Third, and most importantly, the genome regulates all cellular functions, so loss of any portion is catastrophic for a single-celled organism. Ionizing radiation generates multiple types of DNA damage. As much of the damage results from the action of hydroxyl radicals, the spectrum of damage is similar to that produced by oxidative damage associated with endogenous aerobic metabolism. In DNA, the nucleobase is most often modified, and many dozens of different structural modifications to the bases have been characterized. However, 10–20% of the time the sugar-phosphate moiety is affected, which can lead to a single-strand break. The 5′ and 3′ ends at these breaks are usually phosphorylated, indicating that one or more nucleotides have been excised at the break site. The 3′ ends sometimes include a glycolate moiety derived from fragmentation of the deoxyribose. Most of the lesions are accurately repaired by robust DNA-repair systems present in all cells, but double-strand breaks are the most difficult to repair, and therefore the most lethal. They can arise when single-strand breaks occur by chance in close proximity on opposite strands, when a cluster of hydroxyl radicals introduce strand breaks in both strands at one location, or when the cell attempts to enzymatically excise damaged bases present in close proximity on both strands. Double-strand breaks can result in significant loss of genetic information and, if not repaired, will prevent replication of the prokaryotic genome. How an organism deals with double-strand breaks lies at the heart of that organism’s capacity to survive radiation exposure.

High linear energy transfer

– α e

β γ

γ γ

Low linear energy transfer

there is one copy of each gene on each genome copy, Redundant genetic information also functions as N is equal to the number of genome copies). If 100 a reserve that can be used to repair DNA segments inactivating lesions are randomly introduced into that are damaged (or degraded) beyond repair. In a single copy of the D. radiodurans genome (2,897 E. coli K12, repair of DNA double-strand breaks only genes), the probability of inactivating any specific occurs during the exponential growth phase in rich gene on that genome copy is 3.4%. The probability of media, when cells contain multiple chromosomes26, inactivating all the copies of the same gene is reduced and diploid and tetraploid yeast are more resistant to to 0.12% when two genomes are present, and to 0.004% ionizing radiation than isogenic haploid strains37. It has when there are three genomes. been hypothesized that genetic redundancy contributes

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Manganese content. Daly and colleagues examined the effects of the high concentrations of Mn(II) that can accumulate in D. radiodurans27 on the capacity of these cells to survive irradiation. When D. radiodurans cultures are starved of Mn(II), their resistance to ionizing radiation decreases27. The numbers of DNA double-strand breaks formed are the same for a specific dose of ionizing radiation both in the presence or absence of Mn(II), so Mn(II) does not prevent DNA damage27. Instead, cellular damage that results from exposure to high radiation doses is better tolerated if Mn(II) is present. Most DNA damage that occurs after exposure to ionizing radiation results from the generation of reac- 2 µm tive oxygen species (ROS) and the hydrolytic cleavage of water. Intracellular Mn(II) can be protective by Figure 3 | A tetrad of Deinococcus radiodurans. An scavenging ROS. For example, Lactobacillus plantarum epifluorescence image of stationary-phase D. radiodurans R1. lacks the protective enzyme superoxide dismutase, DNA is stained with DAPI and appears blue in the figure; the membrane is stained with the dye FM4-64 and appears red. and instead substitutes with intracellular Mn(II) concentrations of 20–25 mM42,43. As the levels of double- strand breaks in D. radiodurans seem to be unaffected to the radioresistance of Deinococcus spp., although by Mn(II), any scavenging of ROS must protect macro- this idea has not been tested. The number of genome molecules other than DNA. Daly et al.27,44 have pro- copies in D. radiodurans has never been reported to posed that Mn(II) accumulation prevents superoxide be less than four, and cells have the same survival rate and related ROS that are produced during irradiation whether they contain four or ten genome copies23. The from damaging proteins. If this is the case, bacteria contribution of genome redundancy to radioresistance that do not accumulate sufficient Mn(II) might suc- in D. radiodurans (relative to other mechanisms) is cumb to ionizing-radiation-induced protein damage unclear. Many bacteria contain more than one genome before DNA is significantly damaged44. copy, particularly during exponential growth. For Alternatively, the increased Mn(II) concentration example, E. coli contains between five and 18 genome could contribute to the condensation of the D. radio- equivalents during exponential-phase growth38, and durans genome40,45. DNA can be condensed in vitro by Azotobacter vinelandii can accumulate more than 100 adding multivalent cations to an aqueous solution of genome copies39. Neither species is radioresistant, so DNA — the cations neutralize the repulsion of phos- genetic redundancy alone does not account for radio- phate groups in the DNA backbone46. In this way, the resistance. Nevertheless, repair of DNA double-strand proposals of Daly27,44 and Minsky40,45 could be related breaks by recombination requires the presence of more and have similar positive consequences in the context than one genome26,37, so it seems probable that genome of genome reconstitution. redundancy is necessary for genome repair. Regulation of cellular responses to extensive radiation Nucleoid organization. The nucleoids of stationary- damage. When D. radiodurans is exposed to ionizing phase D. radiodurans cells (FIG. 3) are arranged as a radiation, a well characterized sequence of physio- tightly structured ring40 that remains unaltered by logical events take place, including rapid cessation high-dose irradiation41. Minsky and colleagues40 have of DNA replication. At sublethal doses, the duration suggested that this structure passively contributes to of the replication delay always exceeds the time that is D. radiodurans radioresistance by preventing the frag- required to repair the DNA damage that caused inhi- ments that are formed by double-strand breaks from bition of replication47–49. The capacity to inhibit DNA diffusing apart during repair, which maintains the replication is not unlike the DNA-damage checkpoints linear continuity of the genome even when it is frag- of eukaryotes — mechanisms that sense DNA damage mented. Although intellectually appealing, this hypo- and initiate a delay in the cell cycle until the damage is thesis is controversial and has been criticized because repaired50. However, the existence of a DNA-damage the prevalence of ring-like nucleoids under different checkpoint operating in D. radiodurans has not been growth conditions does not always correlate with formally established. ionizing-radiation resistance27,41. A recent examina- tion of the nucleoids of members of the radioresistant Potential enzymatic contributions to repair genera Deinococcus and Rubrobacter revealed a high Ultimately, DNA strand breaks must be enzymatically degree of genome condensation — regardless of nucle- repaired, and Deinococcus spp. can use novel repair oid shape — relative to the more radiosensitive species processes. In 1996, Daly and Minton provided evidence E. coli and Thermus aquaticus 41, which could indicate for a genome-repair process that involved a temporal that species with a condensed genome might be better progression through at least two distinct stages31. protected from ionizing radiation. Substantial chromosome repair occurs during the first

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Table 3 | Selected Deinococcus radiodurans proteins with putative functions in DNA repair Protein Region of homology % identity % similarity Comments Ligase (DnlJ) 11–669/671 Escherichia coli, 42 57 - 29–681/708 D. radiodurans PolA 5–924/928 E. coli, 35 49 - 43–952/965 D. radiodurans PriA 202–729/731 E. coli, 26 42 - 404–922/924 D. radiodurans RecA 4–324/353 E. coli, 57 72 - 16–336/362 D. radiodurans RecD 30–598/609 E. coli, 27 40 N-terminal extension in 218–705/716 D. radiodurans D. radiodurans RecF 3–330/358 E. coli, 28 43 - 6–326/360 D. radiodurans RecG 6–693/694 E. coli, 39 53 N-terminal extension in 107–777/785 D. radiodurans D. radiodurans RecJ 68–570/579 E. coli, 34 51 C-terminal extension in 3–461/685 D. radiodurans D. radiodurans RecN 2–553/553 E. coli, 31 49 - 34–564/564 D. radiodurans RecO 7-157/242 E. coli, 18 34 Low homology; required 10-159/224 D. radiodurans PSI-BLAST RecQ 9–600/608 E. coli, 8–605/824 46, 36, 33 63, 64, 59 HRDC domain repeated D. radiodurans; 557–605/608 E. coli, three times in 680–728/824 D. radiodurans; D. radiodurans 549–606/608 E. coli, 768–825/824 D. radiodurans RecR 1–199/202 E. coli, 42 55 C-terminal extension in 1–196/220 D. radiodurans D. radiodurans RuvA 1–199/203 E. coli, 33 49 - 1–197/201 D. radiodurans RuvB 13–332/337 E. coli, 56 75 A second orthologue with 2–321/333 D. radiodurans weaker similarity is present RuvC 4–168/174 E. coli, 33 51 - 3–166/179 D. radiodurans SbcC 27–1032/1049 E. coli, 21 35 - 22–896/909 D. radiodurans SbcD 1–293/400 E. coli, 28 46 C-terminal changes 24–319/417 D. radiodurans For each protein, the region of homology is indicated as residue numbers, followed by the total number of residues in the protein. The gene for the single-stranded-DNA-binding protein (SSB) is described in the text. No homology found for DnaC, DnaT, PriB, PriC, RecB, RecC, RecE, RecT or SbcB.

1.5 hours after D. radiodurans is treated with a high during recovery from extended desiccation, and 33 of dose of ionizing radiation, through RecA-independent these genes were also induced following irradiation. repair processes. Almost one-third of the double-strand The five genes most highly induced in response to breaks were repaired in this phase. RecA-dependent both stresses were identical and encode proteins of recombinational DNA repair becomes important sev- unknown function. Inactivation of these loci — ddrA, eral hours after irradiation, and predominates in the ddrB, ddrC, ddrD and pprA — produces phenotypes later stages of genome reconstitution31. Proteins that that are relevant for genome repair. Genetic analyses are known to be potentially important in genome- defined three EPISTASIS GROUPS that affect ionizing- repair processes are listed in TABLE 3. radiation resistance, and established that two of the A search for novel genes that are induced in loci (ddrA and ddrB) contribute to radioresistance EPISTASIS GROUP response to ionizing radiation and desiccation, using through different RecA-independent processes. This occurs when two or more genome-based microarrays, provided new evidence for The pprA and recA loci form a third epistasis group, genes control a phenotype. The both RecA-independent and RecA-dependent path- indicating that the pprA gene product interacts with combined effect of mutations in 51 these genes on a phenotype ways of double-strand-break repair . In exponentially RecA. Identification of these novel loci indicates that deviates from the sum of their growing cells, 72 genes are induced three-fold or more there are new mechanisms with an important role in individual effects. after γ-irradiation. Seventy-three loci were induced genome repair of D. radiodurans post-irradiation.

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DNA double strand break Bioinformatics initially failed to identify any DdrA homologues, but further bioinformatics investigation revealed that DdrA was distantly related to the eukaryotic Rad52 protein and to the prokaroytic Redβ, Erf and RecT proteins52,53, so additional functions for End processing DdrA have not been ruled out.

RecA-independent double-strand-break repair. Both NONHOMOLOGOUS END JOINING (NHEJ) and single-strand First strand invasion, annealing (SSA) pathways have been hypothesized to DNA synthesis function in D. radiodurans. Minsky and colleagues40,45 3' suggested that NHEJ would be a useful process for the repair of double-strand breaks in the context of a condensed chromosome, in which ends might not be free to diffuse away from each other. An NHEJ Second strand invasion, 54 DNA synthesis system has been identified in Bacillus subtilis and is probably present in other bacteria. Two other laboratories also recently suggested that NHEJ occurs in D. radiodurans55,56. PprA and PolX are two proteins with predicted activities that are consistent with the existence of NHEJ55,56. However, classical NHEJ sys- Strand separation and tems are generally error-prone57 and seem unsuited annealing to the accurate genome repair that is observed in Deinococcus spp. Patterns of recombination between plasmids and the re-circularization of integrated plasmids in irradiated Deinococcus cells are not consistent Continued DNA synthesis 31 and gap filling with NHEJ . Plasmid repair and re-circularization of genome- integrated plasmids during the RecA-independent Figure 4 | Synthesis-dependent strand annealing. A mechanism of error-free double-strand- phase of DNA double-strand-break repair in break repair that is initiated by creating 3′ overhangs from the ends of the broken DNA duplex (green in the figure). One of these 3′ ends invades a homologous region on an undamaged D. radiodurans is dependent on homology, indicat- 31 sister duplex (blue in the figure), priming DNA synthesis and creating a D-loop that acts as a ing that SSA might have a role . Recent research has template or DNA synthesis primed by the other 3′ end. If displaced, the newly synthesized DNA suggested a compelling model for genome restitu- can anneal, closing the double-strand break. Newly synthesized DNA is coloured red. tion in this species, in which the related but more efficient process called synthesis-dependent single- strand annealing (SDSA) plays a necessary part DNA end protection. The ddrA locus was the first (M. Radman, personal communication). It provides of the five new genes to be characterized52. Deletion evidence that D. radiodurans R1 uses SDSA as a first of ddrA function results in a modest increase in radia- step in genome re-assembly. During SDSA, the 3′ end tion sensitivity in cells grown in rich media. However, of a strand derived from a DNA double-strand break when cells are irradiated and then starved, deletion of invades the homologous region of a sister duplex ddrA results in a 100-fold loss of viability over 5 days (FIG. 4). The invading 3′ end is used to prime DNA compared with wild-type cells. The loss in viability synthesis, unwinding the sister duplex and enlarging of the ddrA mutant is accompanied by a dramatic the D-loop. The displaced strand in the undamaged decrease in genomic DNA content by nucleolytic complex anneals to the remaining free 3′ end cre- degradation. The DdrA protein binds to the 3′ ends ated by the double-strand break. As each 3′ end of single-stranded DNA in vitro and protects them primes complementary DNA synthesis, the result- from nucleolytic degradation52. ing newly synthesized strands can anneal, sealing DdrA seems to function as a DNA-end-protection the breach in the damaged duplex in an error-free NONHOMOLOGOUS END system. As double-strand breaks occur, DdrA (and per- manner. This work shows that all RecA-independent JOINING One of several pathways that haps other proteins) binds to the exposed DNA ends genome assembly requires extensive polA-dependent can be used to repair and prevents nuclease digestion of the chromosomal DNA synthesis, and that the pattern of nucleotide chromosomal double-strand DNA. This strategy is particularly useful in the genome in corporation, as measured by density labelling DNA breaks. The process is repair that occurs after desiccation. DNA repair uses a post-irradiation, indicates a distributive mode of non-homologous because adjacent broken strands are lot of metabolic energy, but cells recovering after desic- replication that is consistent with SDSA. The amount fused by direct end-to-end cation in an environment that lacks nutrients would not of newly synthesized DNA recorded during this contact without regard to have the opportunity to repair DNA damage. As DNA phase of post-irradiation recovery indicates that the sequence homology. Therefore, strand breaks occur, nuclease action could degrade tails are longer than expected based on precedent in non-homologous end joining is error-prone because it results in genomic DNA. By protecting the broken DNA ends, other species that exhibit SDSA. The authors suggest joining of the breaks without a cells could preserve genomic DNA until conditions that the long tails assure precise annealing, ensuring template. become suitable for cell growth and DNA repair. an error-free recovery.

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E. coli RecA D. radiodurans RecA Based on the properties of an E142K mutant of Single-stranded DNA Double-stranded DNA D. radiodurans RecA (also known as RecA424), Satoh et al.60 suggested that the effect of D. radiodurans RecA on double-strand-break repair primarily reflected a RecA RecA regulatory rather than a recombination function. The mutant protein does not complement the null phenotype of a recA knockout in E. coli, but does have residual regulatory functions associated with RecA. Strains har- Double-stranded DNA Single-stranded DNA bouring the E142K mutation seem to retain resistance to γ-irradiation. However, the E142K mutation does retain significant DNA-strand-exchange activity in some assays, and more work is needed to define the function of RecA in D. radiodurans. Assuming that D. radiodurans + + must locate and splice together overlapping segments of its chromosomes to reconstruct a functional genome, Figure 5 | Inverse DNA-strand exchange promoted by the Deinococcus radiodurans a DNA-pairing activity such as that provided by RecA RecA protein. Instead of forming a filament on single-stranded DNA and then reacting with a homologous duplex, strand exchange is initiated by a filament formed on duplex DNA. would be at the centre of such a process. The ssb gene in D. radiodurans was originally annotated as a tripartite gene, with two frameshifts required to form a functional open reading frame At least one additional RecA-independent path- (ORF) for translation20,61–63. However, this locus seems way mediated by the DdrB protein is present in to be specific to the D. radiodurans R1 strain that was D. radiodurans51. Although DdrB has not yet been char- sequenced (ATCC BAA-816), as it differs substantially acterized in vitro, deletion of ddrA and ddrB produces from that of the D. radiodurans R1 type strain (ATCC a mutant that is significantly more sensitive to ionizing 13939). In the type strain, the ssb locus is a single radiation than either ddrA or ddrB mutants, indicating continuous ORF that encodes the largest bacterial that DdrA and DdrB have complementary activities. SSB polypeptide identified to date. It has two oligo- nucleotide/oligosaccharide-binding (OB) folds rather Recombinational DNA repair. Any ends that lack than the one present in most bacterial SSB proteins, overlapping sequence or other means to guide accurate and it functions as a dimer rather than a tetramer64. repair require recombinational repair using redundant The gene is highly homologous to ssb genes found in genome information. Studies of the roles of the clas- Thermus species, to which D. radiodurans is closely sical proteins involved in bacterial recombinational related. The D. radiodurans SSB protein is efficient at DNA repair in D. radiodurans have been initiated stimulating the DNA-strand exchange that is promoted TABLE 3, including studies of recombinase A (RecA), by RecA proteins from both E. coli and D. radiodurans, single-strand-binding protein (SSB), recombinase D being more active than the E. coli SSB in both cases. (RecD), DNA polymerase I, recombinase R (RecR) The atomic structure of D. radiodurans SSB has been and recombinase O (RecO). solved65. This protein could be important in chromo- The D. radiodurans RecA protein (361 amino acids, some repair. The concentration of the D. radiodurans

Mr 38,013) is 57% identical (72% similar) to the E. coli SSB is an order of magnitude higher than the normal

RecA protein (352 amino acids, Mr 37,842). In vitro, the in vivo levels of the SSB protein in E. coli. protein promotes all of the key recombinogenic activities Less is known about other classical repair proteins in of RecA-class recombinases. It forms filaments on DNA, D. radiodurans. In the absence of a recB or recC gene, the hydrolyses ATP and dATP in a DNA-dependent fashion recD gene product has been characterized. The D. radio- and promotes DNA-strand exchange58. However, the durans RecD protein has a 5′ to 3′ helicase activity, in D. radiodurans RecA protein has one distinct function. common with the RecD subunit of the E. coli RecBCD The DNA strand-exchange reactions of the E. coli RecA enzyme66. It can unwind short (20 bp) duplexes if a protein, and all other homologues examined to date, are 5′ single-strand tail is adjacent. The atomic structure ordered so that the single-stranded DNA is generally of the D. radiodurans RecO protein has been solved67, bound first, before the double-stranded DNA is bound. but nothing is known about its function. Similarly, the This order of DNA binding makes sense from a biologi- structure of the Deinococcus RecR protein has been cal standpoint, as the RecA protein must be targeted to determined68. single-strand gaps at stalled replication forks and other Narumi and colleagues have shown that purified damaged DNA sites. By contrast, the D. radiodurans PprA protein can bind to duplex DNA with strand RecA protein promotes an obligate inverse DNA- breaks in vitro, protect the strands from nuclease strand-exchange reaction59, binding the duplex DNA digestion and facilitate the ligation of duplex DNA first and the homologous single-stranded DNA sub- fragments56. They speculate that PprA is part of a deino- strate second (FIG. 5). It is probable that this reaction coccal NHEJ system, although this role seems incon- pathway reflects the function of D. radiodurans RecA sistent with the absence of mutations observed after in double-strand-break repair, although its significance irradiation in this species. As with most D. radiodurans is not yet clear. proteins, PprA characterization is at an early stage.

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DSBs in a typical cell Proposed repair of cryptic DSBs in scored as double-strand breaks during the processing Deinococcus radiodurans IR and analysis of genomic DNA because the conditions necessary to stabilize the paired single-strand breaks •OH •OH are lost when the cell is disrupted. However, in situ the DNA strands are not separated. Unlike classical NHEJ, •OH •OH repair of these cryptic breaks would be effectively tem- plated, as genomic continuity would never be lost. This proposal envisions a specialized system operating in the condensed chromosome that prevents the formation of double-strand breaks at those sites where DSB avoidance through constraint by proteins some base pairing is present. Opposed single-strand or conditions breaks would be repaired using a set of enzymes that could deal with the close proximity of the breaks in opposing strands without affecting this base pairing. By this mechanism, the linear continuity of the genome Patch/repair sequence, including regions rich in short nucleotide repeats, would be preserved at many potential sites of Repair or cell death double-strand breaks in a manner that is error-free. Experimentally, this process would be indistinguish- Figure 6 | A proposed mechanism that might contribute to the tolerance of radiation damage in Deinococcus radiodurans. Potential double-strand breaks (DSBs) able from a non-mutagenic type of NHEJ. If such a are constrained, held together by proteins and/or their local environment, so that many system exists, the repair would be accurate and RecA- breaks scored as DSBs in in vitro assays are cryptic in the cell. IR, ionizing radiation; independent, passively reducing the cell’s dependence •OH, hydroxyl radical. on recombinational DNA repair and the accompanying homology search at any break site that is stabilized in this manner. A new proposal for radiation tolerance Examples from the literature indicate that the cell To the list above, we add one more mechanism, which might use two alternative, but not mutually exclu- is a new proposal (FIG. 6). It is particularly difficult to sive, mechanisms to stabilize base pairing between explain the accurate repair of the hundreds of scattered opposed single-strand breaks. First, D. radiodurans repeat elements in the D. radiodurans genome20,63,69 might encode proteins that hold the DNA together. after the introduction of hundreds of double-strand Second, the intracellular ionic composition could breaks. How does D. radiodurans avoid the potentially be sufficient to physically limit dissociation of DNA disruptive recombination events that could occur base pairs. If proteins are responsible, we assume that between these repeated elements to maintain the con- they will be functionally analogous to the structural- tinuity of its genome? We speculate that D. radiodurans maintenance-of-chromosomes (SMC) proteins that uses a strategy that potentially combines elements of are present in many eukaryotic and prokaryotic spe- many of the systems described in this review to avoid cies70–72. In eukaryotes, these proteins are referred to this problem — including the condensed nucleoid as cohesins and condensins, and the significance of structure first highlighted by Minsky40,45 and the high their role in genome stabilization and DNA repair is concentrations of Mn(II) ions discussed by Daly27,44 becoming apparent73. The stability of annealed comple- — thereby improving the cell’s capacity to tolerate mentary DNA is dependent on the ionic strength of the ionizing radiation. medium in which the DNA is dissolved, and increased Many DNA double-strand breaks arise from the intracellular Mn(II) concentrations27 might help to juxtaposition of two single-strand breaks that form hold DNA that contains several single-strand breaks as a function of the distance in base pairs between together. Freifelder and Trumbo74 have shown that these breaks BOX 1. In general, the further apart the high-ionic-strength media stabilize opposed breaks single-strand breaks, the less likely it is that the DNA separated by as little as two base pairs ends will separate to form a double-strand break. We A system for the repair of cryptic double-strand suggest that a significant contribution to the observed breaks might exist in many organisms. Conceptually, tolerance of ionizing radiation in D. radiodurans could the damage that is caused by high-dose ionizing arise if many of the measured double-strand breaks radiation is similar to the damage that would occur that occur are cryptic in vivo. In other words, some if a type II restriction enzyme was expressed in vivo. fraction of the measured double-strand breaks could A large number of opposed single-strand breaks actually be held together so that the separation of the would be formed and would produce double-strand DNA ends never really occurs in the cell. This pro- breaks unless stabilized. In yeast, the prolonged posal assumes that the organism has a mechanism to expression of EcoRI produces thousands of breaks stabilize opposed breaks, constraining the intervening but results in a surprisingly modest (2–3 fold) loss of base pairs so that actual separation of the two DNA viability75–77. Similarly, prolonged artificial expression ends does not occur. This idea is distinct from proof the yeast mating-type-specific HO endonuclease posals40,45 in which the ends separate and are repaired results in only a 35% loss of viability78 in strains that by NHEJ. The cryptic breaks we propose would be have four HO-sensitive chromosomal loci. Survival

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in these strains does not depend on homologous- Our interpretation of the literature published to recombination systems. Lethal fragmentation of date on the Deinococcaceae suggests no extraordinary chromosomes only occurs in cells that have defective survival strategies, or at least no single ‘magic bullet’ NHEJ or checkpoint-control systems. Even E. coli that provides a complete explanation for the phenom- cells are surprisingly resistant to EcoRI-mediated enon. Instead, enhanced radioresistance seems to be chromosomal cleavage as long as the appropriate the consequence of an evolutionary process that has DNA ligases are not inactivated79. Once the cryptic coordinated various passive and active mechanisms, breaks are repaired, breaks where the ends have sepa- enabling survival from stresses such as desiccation. rated could be repaired by additional processes such The almost ubiquitous classical DNA-repair pathways, as SSA and recombinational DNA repair. perhaps with some specialized properties, seem to be augmented by novel features of deinococcal nucleoid Conclusion structure and metabolism. It is currently difficult to The extraordinary phenotypes of D. radiodurans predict which mechanism(s) will be most important in have encouraged a host of rather fanciful descrip- radioresistance, or even whether all of the contributing tions of the origin of this organism. Stories of the mechanisms have been discovered. Each of the many arrival of this species on earth on a comet, or arising enhancements could individually have a modest effect, through mutations owing to mankind’s attempts to but collectively they mediate radioresistance. These harness nuclear power, are readily available on the changes need not be unique to D. radiodurans, but internet (See Online links box). These ideas are likely might be present in other organisms. to flourish until this remarkable organism is better Therefore, the capacity of the deinococci to deal with understood. D. radiodurans has a readily documented stresses that cause massive genetic damage might not be evolutionary origin within the domain Bacteria. It is as strange or as unusual as it once seemed. The combi- clearly related to the rest of life on this planet, carrying nation of repair strategies is sophisticated and effective, out all of the fundamental processes that have been but the individual strategies delineated so far are based characterized in prokaryotes. on mechanisms present in many other organisms.

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